1 Tissue Injury and Repair Group, Research School of Clinical and Laboratory Sciences, Faculty of Medical and Human Sciences, Stopford Building, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK

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Abstract

Introduction

Chronic and debilitating low back pain is a common condition and a huge economic burden.
Many cases are attributed to age-related degeneration of the intervertebral disc (IVD);
however, age-related degeneration appears to occur at an accelerated rate in some
individuals. We have previously demonstrated biomarkers of cellular senescence within
the human IVD and suggested a role for senescence in IVD degeneration. Senescence
occurs with ageing but can also occur prematurely in response to stress. We hypothesised
that stress-induced premature senescence (SIPS) occurs within the IVD and here we
have investigated the expression and production of caveolin-1, a protein that has
been shown previously to be upregulated in SIPS.

Results

Caveolin-1 gene expression and protein expression were demonstrated within the human
IVD for the first time. NP cells from degenerate discs exhibited elevated levels of
caveolin-1 which did not relate to increasing chronological age. A negative correlation
was observed between gene expression for caveolin-1 and donor age, and no correlation
was found between caveolin-1 protein expression and age. A positive correlation was
identified between gene expression of caveolin-1 and p16INK4a.

Conclusion

Our findings are consistent with a role for caveolin-1 in degenerative rather than
age-induced changes in the NP. Its expression in IVD tissue and its association with
the senescent phenotype suggest that caveolin-1 and SIPS may play a prominent role
in the pathogenesis of IVD degeneration.

Introduction

Low back pain (LBP) is a condition that affects a significant proportion of the population,
with a lifetime incidence rate in excess of 70% in industrialised nations [1]. It not only impacts on quality of life, but also places a substantial financial
burden on the National Health Service and the economy in general due to loss of working
days [1,2]. Many cases of LBP are attributed to degeneration of the intervertebral disc (IVD)
and imaging studies have indicated a link between IVD degeneration and LBP [3,4].

To date, no clear mechanism for IVD degeneration has been identified, although the
involvement of both environmental and genetic factors has been proposed [5-8]. The occurrence of IVD degeneration increases with age [9,10]; however, a subset of individuals appear to exhibit accelerated degeneration that
is independent of age [5,6]. This has led to speculation that additional factors could play a key role in the
development of degeneration in some individuals.

There is increasing evidence that many features of IVD degeneration, including altered
matrix synthesis and enhanced matrix degradation, originate at a cellular level [6,11,12]. Cellular senescence is a strong candidate for the prolonged alteration in cellular
activity observed during degeneration. Senescence and accompanying alterations in
cell function have been implicated in ageing-related, degenerative, and pathological
changes in a variety of tissues, including atherosclerotic plaque development within
blood vessels and osteoarthritic alterations to cartilage [13-15]. Two groups have shown increased staining for senescence-associated β-galactosidase
(SA-β-gal) in cells from prolapsed and degenerate IVD cells, respectively, when compared
with non-degenerate discs [16,17]. More recently, our group has presented more comprehensive evidence of senescence
biomarkers in human IVD samples, demonstrating increased cellular senescence during
IVD degeneration [18]. In particular, cells from degenerate discs exhibited increased SA-β-gal activity,
elevated expression of the cell cycle inhibitor p16INK4a, telomere erosion, and a decrease in replicative potential. Furthermore, a correlation
was observed between p16INK4a expression and the expression of matrix-degrading enzymes matrix metalloproteinase
(MMP)-13 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5,
suggesting a role for cell senescence in the molecular processes observed during IVD
degeneration [18].

Senescence occurs naturally with ageing but can also occur prematurely in response
to stresses (such as exposure to cytokines or oxidative stress) in a number of cell
types [19-24]. Since telomeric erosion and p16INK4a protein expression are increased in degenerate discs compared to non-degenerate age-matched
samples [18], we hypothesised that stress-induced premature senescence (SIPS) occurs within the
IVD and may be responsible for the accelerated degeneration observed in some individuals.

Caveolae are plasma membrane compartments found abundantly in terminally differentiated
cells such as fibroblasts and endothelial and muscle cells [25]. The mammalian caveolin gene family codes for three 21 to 25 kDa caveolin proteins,
which are integral membrane proteins essential for the structural integrity and function
of caveolae [26]. Expression of caveolin-3 is muscle-specific, whereas caveolin-1 and caveolin-2 are
coexpressed in many cell types [26]. Proposed functions include lipid transport, membrane trafficking, and a role in
intracellular signalling pathways which stems from the colocalisation of caveolins
with a variety of signal transduction molecules [25-28]. Interestingly, caveolin-1 has been implicated in the senescent phenotype of several
cell types, including human fibroblasts, lung adenocarcinoma cells, endothelial cells,
and articular chondrocytes [19,29-33]. Moreover, caveolin-1 has been proposed to mediate SIPS in murine fibroblasts and
human articular chondrocytes in response to oxidative stress and the inflammatory
cytokine interleukin-1β (IL-1β) (both of which are known to be increased during IVD
degeneration) [19,31,34-38]. Here, we have investigated the expression of caveolin-1 in human IVDs and correlated
its expression with the cell cycle inhibitor and the biomarker of senescence p16INK4a, focusing on the nucleus pulposus (NP) as this area shows the most evidence of cell
senescence in human IVDs [18].

Materials and methods

Tissue samples

Human IVD tissue was obtained either at post mortem (PM) examination or from patients
undergoing surgery, where patients were selected on the basis of magnetic resonance
imaging-diagnosed degeneration and progression to anterior resection either for spinal
fusion or disc replacement surgery for chronic LBP. Local research ethics committee
approval was obtained together with informed consent from the patient or relatives.
Disc tissue was removed as detailed previously [37].

General procedure for tissue specimens

A block of tissue (incorporating annulus fibrosus [AF] and NP in continuity) was fixed
in 10% vol/vol neutral buffered formalin and embedded in paraffin wax. Four micron
sections were stained with haematoxylin and eosin to grade the degree of morphological
degeneration according to previously published criteria that assess the demarcation
between NP and AF, proteoglycan content of the NP, presence and extent of structural
fissures, and cell cluster formation [39]. Potential grades range between 0 and 12. A grade of 0 to 3 indicates a histologically
non-degenerate IVD, 4 to 7 indicates evidence of intermediate (or moderate) degeneration,
and 8 to 12 indicates severe degeneration. Further tissue sections were taken for
immunohistochemical analysis of caveolin-1.

Conventional reverse transcription-polymerase chain reaction

To investigate gene expression of caveolin-1 in human NP cells, RNA was extracted
from isolated cells following the standard procedure for TRIzol® reagent (Invitrogen). cDNA was then synthesised using Superscript II in accordance
with the instructions of the manufacturer (Invitrogen). A standard Platinum Taq (Invitrogen)
method was used for conventional polymerase chain reaction (PCR), using a concentration
of 1.5 mM MgCl2. Primers specific for caveolin-1 [19] and the housekeeping gene 18S (Invitrogen) are detailed in Table 1. All primers were confirmed for gene specificity using BLAST (Basic Local Alignment
Search Tool) (Genbank database sequences). Reactions, including non-template controls,
were conducted for 35 cycles, including the annealing temperature of 58°C on a thermal
cycler (MJ Research, now part of Bio-Rad Laboratories, Hercules, CA, USA), and products
were analysed alongside a 100-base pair DNA ladder (Hyperladder IV; Bioline, London,
UK) by electrophoresis on a 1.5% wt/vol agarose gel containing 0.2 μg/mL ethidium
bromide (Sigma-Aldrich). Product bands were visualised by UV transillumination and
images were captured using Gene Snap software (Syngene, Cambridge, UK).

Quantitative real-time polymerase chain reaction

Quantitative real-time reverse transcription-PCR (qRT-PCR) was performed to further
examine caveolin-1 gene expression in human NP cells and to investigate any correlation
between caveolin-1 and p16INK4a gene expression in isolated NP cells using the standard curve method of analysis as
described previously [18].

Primers and probe design

Primers and FAM-MGB probe specific for human caveolin-1 were designed by Applied Biosystems
(ABI) (Warrington, UK) upon provision of caveolin-1-specific exon sequence (Gene expression
assays) (Table 1). p16INK4a primers and probe were as described previously [18], and 18S primer/VIC-TAMRA probe set was a pre-developed assay reagent (PDAR) purchased from
ABI.

Genomic curve standards

Genomic DNA (gDNA) was used to create standard curves for absolute quantification
of copy number per reaction. gDNA (Promega Corporation, Southampton, UK) was homogenised,
diluted to 100 ng/μL, and sonicated on ice. Serial dilutions of gDNA were prepared
to generate standards with gene copy numbers of 75,000, 7,500, 750, 75, and 0 copies
per 25 μL reaction.

qRT-PCRs were carried out in triplicate in a 96-well plate. Reactions contained 12.5
μL of mastermix (Taqman® Universal PCR mastermix; ABI) and 2.5 μL of template cDNA or gDNA. Primers were added
to a final concentration of 900 nM and probe to a concentration of 250 nM, and molecular-grade
water was added to a total reaction volume of 25 μL. A gDNA standard curve for each
gene was included on each plate. Real-time PCR was performed using an ABI Prism 7000
sequence detection system (ABI). Reactions consisted of an initial Taq activation
step of 95°C for 10 minutes to denature DNA and activate Taq polymerase followed by
40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

Following amplification, an auto-baseline was set using the ABI 7000 sequence detection
software and a threshold was set for each gene, above background levels and within
the exponential phase. From these, a cycle threshold (Ct) was obtained for each well
and data exported into Microsoft Excel (Microsoft Corporation, Redmond, WA, USA),
where the three Ct values for each sample were averaged. Data were analysed as described
previously [18] and results were expressed as copy number of target gene per 100 ng cDNA normalised
to 18S.

Sections were visualised using a Leica RMDB microscope (Leica Camera Limited, Knowlhill,
Milton Keynes, UK), and images were captured using a digital camera and Bioquant Nova
image analysis system (Bioquant Image Analysis Corporation, Nashville, TN, USA). For
analysis, the NP was identified morphologically within each disc section. Within each
section, a minimum of 200 NP cells were analysed from at least five different fields
of view and immunopositivity was calculated as a percentage of the total cell population.

Statistical analysis

Data were non-parametric and thus Mann-Whitney U tests were conducted to compare gene copy number and numbers of caveolin-1-immunopositive
cells in non-degenerate NP (grades 0 to 3) and degenerate NP (grades 4 to 7 and 8
to 12). Non-parametric linear regression analysis was performed to analyse the correlation
between copy numbers of different genes and between gene copy numbers and subject
age or number of caveolin-1-immunopositive cells and subject age.

Results

Caveolin-1 gene expression in human nucleus pulposus cells

cDNAs derived from cells directly extracted from the NP of 19 different IVDs, from
both PM and surgical sources, were analysed for expression of the caveolin-1 gene.
Eight samples were taken from non-degenerate IVD (grades 0 to 3; mean age ± standard
deviation [SD] 45.4 ± 18.7 years) and 11 samples from degenerate IVD (grades 4 to
9; 51.7 ± 24.3 years). Gene expression for caveolin-1 was detected in the NP tissue
of every sample analysed (qRT-PCR analysis). Comparison of caveolin-1 gene expression
by non-degenerate and degenerate samples demonstrated higher gene expression in degenerate
samples (conventional RT-PCR analysis, Figure 1). This was supported by qRT-PCR analysis (Figure 2a) in that non-degenerate samples demonstrated a median caveolin-1 gene copy number
of 35,220 with a range of 6,740 to 70,9222 copies per 100 ng cDNA compared with the
elevated degenerate median caveolin-1 gene copy number of 45,695 with a range of 7,589
to 105,626 copies per 100 ng cDNA (Figure 2a). A negative correlation was observed between gene expression for caveolin-1 and
age of the donor (P = 0.0472) (Figure 2b).

Caveolin-1 protein expression was investigated in 28 IVD samples (for sample details,
see Table 2). Immunohistochemical analysis for caveolin-1 demonstrated cytoplasmic/membrane staining
within the chondrocyte-like cells of the NP (Figure 3). The percentage of immunopositive cells for caveolin-1 increased from 2.59% ± 1.01%
(mean ± standard error of the mean [SEM]) in non-degenerate discs to 13.62% ± 6.51%
in severely degenerate samples (Figure 4a). All IgG1 controls were negative. It must be noted that the majority of patients
with severely degenerate discs were above 50 years of age; however, in the 24 samples
of all grades for which the chronological age of individuals was known, no correlation
was observed between caveolin-1 immunopositivity and age of the donors (P = 0.6609) (Figure 4b).

Discussion

This study has demonstrated for the first time that cells from the NP of human IVDs
express caveolin-1 and furthermore that caveolin-1 gene expression and protein expression
are elevated in degenerate IVDs, but that this rise in caveolin-1 expression does
not correlate with increasing age. This is consistent with a role for caveolin-1 in
degenerative rather than age-induced changes in the NP.

Changes associated with tissue ageing and degeneration have been postulated to involve
cellular senescence [41-43]. Two major categories of senescence are generally described in the literature as
replicative senescence (RS) and SIPS. RS was first described by Hayflick in 1965 [44] and is widely regarded as one of the main mechanisms underlying the normal ageing
process via reduction of telomere length to critical levels following cumulative population
doublings. In addition, there are a number of reports describing premature induction
of senescence as a result of cellular exposure to stress. Factors linked to the induction
of SIPS vary widely, from DNA damage – for example, radiation (bovine aortic endothelial
cells [45]), UV light (human fibroblasts [46] and human melanocytes [47]), chemical treatment (nasopharyngeal carcinoma cells [48] and human fibroblasts [49,50]), and oxidative stress (human fibroblasts [20,22,24] and human articular chondrocytes [19]) – to oncogenic protein overexpression (for example, ras in human fibroblasts [51]) and exposure to inflammatory cytokines such as IL-1 and tumour necrosis factor-α
(human chondrocytes and fibroblasts [19,21,23]). Previous data from our laboratory described accelerated senescence (characterised
by a variety of biomarkers, including reduced cell replication potential, elevated
levels of the cell cycle inhibitor p16INK4a, increased SA-β-gal activity, and telomere erosion) in degenerate human IVDs compared
with age-matched non-degenerate discs [18], suggesting that SIPS may be involved in IVD degeneration.

Caveolin-1 forms homodimers, or heterodimers with its family member caveolin-2, that
insert into the plasma membrane of terminally differentiated cells [25]. The caveolin-1-rich areas termed caveolae and the caveolin proteins themselves are
proposed to regulate cellular processes, including membrane traffic, signal transduction,
and cellular senescence [25-28,52]. Caveolin-1 was investigated here due to its possible role in cellular senescence,
in particular SIPS [19,31,52]. Here, we show that caveolin-1 gene expression and protein expression are increased
during IVD degeneration, but not in a manner that is associated with increasing chronological
age.

Moreover, we demonstrate a correlation between caveolin-1 and p16INK4a gene expression. p16INK4a is a cyclin-dependent kinase inhibitor that prevents retinoblastoma phosphorylation
and arrests the cell cycle in the G0/G1 phase prior to entry into the synthesis phase [53,54]. Many studies have shown increased levels of p16INK4a alongside the occurrence and maintenance of permanent growth arrest and senescence,
including a rodent model of ageing [55-57]. Previous studies by our group and others strongly suggest a role for p16INK4a in cellular senescence within degenerate tissue when compared with age-matched controls
[18,58]. Furthermore, elevated p16INK4a expression has been described in the premature senescence of human fibroblasts and
leukaemic cells exposed to oncogenic ras and DNA double-strand breaks [51,59,60], strengthening the reports that p16INK4a is a biological marker for senescence. The present study demonstrated that the increased
expression of caveolin-1 seen in the degenerate NP positively correlated with gene
expression for p16INK4a, suggesting that caveolin-1 expression is linked to the senescent phenotype observed
in these cells.

The literature describes evidence linking cell exposure to stressful stimuli to both
caveolin-1 expression and cellular senescence. In mouse NIH 3T3 fibroblasts, administration
of subcytotoxic levels of H2O2 to experimentally mimic oxidative stress induced cellular senescence and increased
caveolin-1 expression. Treatment with H2O2 in the presence of caveolin-1 antisense oligonucleotides reduced expression of senescence
biomarkers, whereas transgenic overexpression of caveolin-1 induced SIPS [31]. In human endothelial cells, isolated from atherosclerotic patients and induced to
senesce, caveolin-1 expression was correlated with senescence biomarkers and with
expression of 4-hydroxynonenal expression (a marker of lipid peroxidation and thus
oxidative stress) independently of an effect on telomere length [31]. These studies strongly support a role for caveolin-1 in SIPS induced by oxidative
stress and this is further strengthened by work conducted on osteoarthritic articular
chondrocytes. Administration of H2O2 to these chondrocytes induced cellular senescence via expression of the caveolin-1
protein, a mechanism reversed by antisense oligonucleotide-mediated downregulation
of the caveolin-1 gene [19]. The same study demonstrated an identical role for the inflammatory cytokine IL-1β.

Articular chondrocytes and the degenerative process observed during osteoarthritis
share many characteristics with IVD cells and IVD degeneration [12,43]. Interestingly, IVD cells are subjected to both oxidative stress and catabolic cytokines,
which have been implicated in the induction of SIPS [19-22,24]. Work published by our group suggests that IL-1β not only is increased in degenerate
discs but is an important factor involved in catabolic events during IVD degeneration,
including decreased matrix production and increased MMP and ADAMTS expression [37,38,61,62]. Moreover, advanced glycation endproducts (AGEs) such as carboxymethyl-lysine (CML)
and the receptor for AGEs (RAGE) have been localised to the NP of degenerate IVD [34-36]. CML is a tissue marker for accumulated oxidative stress [35]; therefore, its presence and that of its receptor RAGE are highly significant for
both mechanisms underlying IVD degeneration and the likelihood that they could cause
SIPS in human NP cells. Furthermore, RAGE has been localised to caveolin-1-rich membranes
in endothelial cells [63]. This gives evidence, together with studies involving IL-1, that there are factors
in the degenerate disc that may induce caveolin-1 expression and thus lead to the
senescent phenotype described in IVD cells [16-18].

Caveolin-1-rich regions of the plasma membrane have been associated with several receptors
and signalling molecules, predominantly through isolation of caveolae and colocalisation
studies. These studies have highlighted a subset of proteins that are relevant to
IVD degeneration and to SIPS. First, RAGE, described above, is known to regulate several
intracellular signalling pathways, including the nuclear factor-kappa-B pathway, which
is essential for the expression of MMPs present in the degenerate IVD [34,64]. Second, there is evidence suggesting that caveolin-1, β1 integrin, and urokinase
plasminogen activator receptor (uPAR) colocalise in human articular chodrocytes [65]. uPAR has an integral role in plasmin activation and thereby promotes catabolic events
through initiation of a proteolytic cascade through which matrix-degrading enzymes
described in IVD degeneration such as MMPs are activated [66]. Both could conceivably be pathways via which elevated caveolin-1 levels exert aspects
of the senescent cellular phenotype observed in IVD degeneration.

Conclusion

This study has shown that caveolin-1 expression in human NP cells is linked to IVD
degeneration and is associated with the senescent phenotype as depicted by increased
expression of p16INK4a. Caveolin-1 expression was not linked to increasing chronological age, suggesting
a role in accelerated degeneration which could be due to SIPS, rather than RS. Further
work will elucidate the role of caveolin-1 in these related areas.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SKH participated in the design of the study, performed the majority of the laboratory
work and analysis, and drafted the manuscript. CLM helped to secure funding, participated
in the design of the study and the interpretation of data, and assisted in the preparation
of the final manuscript. JAH conceived the study, secured funding, contributed to
the design and coordination of the study, and participated in the interpretation of
data and extensive preparation of the final manuscript. All authors read and approved
the final manuscript.

Acknowledgements

This work was funded by a grant from DISCS (Diagnostic Investigation of Spinal Conditions
and Sciatica) and was undertaken in the Human Tissue Profiling Laboratories of the
Tissue Injury and Repair research group.